The applicant has submitted this proposal to develop a foundation in the molecular biology of aging, with a focus on the regulation of heat shock gene expression. Heat shock genes are normally expressed as part of the cellular stress response to unfavorable conditions. These conditions are approximated clinically by ischemia, inflammation, and infection, all which are disproportionately represented in the elderly. Thus, age-dependent changes in the cellular stress response could lead to a decreased ability of the aged to recover from physiologic stress and disease. In phase I, it is hypothesized that aging cells accumulate damage from a life-time exposure to free-radical and oxidative injury, which in turn, leads to a constitutive expression of the heat shock genes. The resultant increase in basal heat shock protein levels would limit further induction of the heat shock genes during future stress, thereby increasing cellular vulnerability to injury and death. To test this hypothesis, this project is designed to characterize the affect of age on the molecular switches regulating the cellular stress response. Specifically, transcriptional regulation of the heat shock genes during clonal senescence of IMR-90 cells will be analyzed. Heat shock transcription factor (HSF) binding activity in senescent cells will be assessed, with the expectation that basal levels of HSF binding increase and inducible levels decrease during senescence. Changes in HSF binding activity will be further characterized by examining HSF gene expression, protein levels and post-translational modifications. By establishing a molecular mechanism for an altered stress response during senescence, the next phase of this proposal will be to establish the significance of an altered stress response in the human population. One approach to this problem will be to determine how cells lose and regain their growth potential during stress at different phases of the cell cycle. Here the affect of stress on growth-associated signals and gene expression will be examined in peripheral blood lymphocytes. The goal will be to extend these findings to lymphocytes from elderly subjects, whose increased sensitivity to the untoward affects of stress could reveal a new dimension of immunosenescence.